Method for decreasing interference in results of immunochemical methods

Abstract

Methods and materials are provided for decreasing or eliminating interference from molecules resulting from upstream immunochemical procedures, such as Immunoprecipitation, that employ an Immunoglobulin Binding Molecule (an IBM, e.g., Protein A) in subsequent downstream methods, such as Western Blot. An effective amount of a ligand of the IBM is used to block the IBM or fragments of the IBM on a support membrane used in the downstream method. In another embodiment, a secondary antibody that is specific either for heavy chain or light chain antibody fragments or that recognizes intact antibody molecules but does not recognize individual antibody fragments may be employed with the ligand of the IBM.

1 . In an immunochemical analytical method having an upstream procedure that employs an immunoglobulin binding molecule (IBM) to produce a product of the upstream procedure and a downstream procedure to treat the product of the upstream procedure, the step of adding a ligand of the IBM to block a binding site of the IBM as a part of the downstream procedure.  
   
   
       2 . The method of  claim 1  wherein the upstream procedure is immunoprecipitation or immunoaffinity purification.  
   
   
       3 . The method of  claim 1  wherein the downstream procedure includes the step of bonding at least one component of the product of the upstream procedure to a solid support.  
   
   
       4 . The method of  claim 1  wherein the downstream procedure is Western Blot or Dot Immunoblotting.  
   
   
       5 . The method of  claim 1  wherein the IBM comprises Protein-A or Protein-G.  
   
   
       6 . The method of  claim 1  wherein the IBM is selected by a method that determines binding to immunoglobulins.  
   
   
       7 . The method of  claim 1  wherein the IBM is identified by analysis of materials selected from a library of synthetic compounds, an engineered protein, a compound made in a mammalian system, a polymer made in a non-mammalian system, a compound prepared by recombinant DNA technology, a polymer made by  E. coli  phage display, a protein scaffold library, a peptide display library, a directed evolution library, or a protein array-based library.  
   
   
       8 . The method of  claim 1  wherein the ligand comprises a molecule that occupies the immunoglobulin binding site of an IBM or fragments thereof.  
   
   
       9 . The method of  claim 1  wherein the ligand is isolated from animals.  
   
   
       10 . The method of  claim 9  wherein the ligand comprises pig IgG.  
   
   
       11 . The method of  claim 1  wherein the ligand is selected from the group of compounds consisting of an immunoglobulin, a portion of an immunoglobulin, a fragment of an immunoglobulin, a variant of an immunoglobulin, an engineered protein, a polymer scaffold, an engineered compound, a polypeptide, a compound made in a mammalian system, a polymer made in a non-mammalian system, and a polymer made in an  E. coli  phage display.  
   
   
       12 . The method of  claim 1  wherein the downstream procedure includes use of a primary antibody and a secondary antibody or IBM, the secondary antibody or IBM having a reporter molecule, further comprising the step of selecting the secondary antibody or IBM to be responsive to only a selected component of the product of the upstream procedure.  
   
   
       13 . A method of analysis for a protein solution having a protein of interest, comprising: 
 a) adding an immunoprecipitating antibody, an immunoglobulin binding molecule and a solid support to the protein solution;    b) separating the solid support from the supernatant solution and dissociating the components of the solid support;    c) separating the dissociated components by electrophoresis;    d) transferring the separated components to a support membrane;    e) placing the support membrane in a solution of an effective amount of a protein to saturate the unreacted protein binding sites on the support membrane and an effective amount of a ligand of the immunoglobulin binding molecule to block any immunoglobulin binding molecule or fragments thereof;    f) placing the support membrane in a solution of a selected primary antibody;    g) transferring the support membrane to a solution of a selected secondary antibody having a reporter molecule thereon; and    h) developing the support membrane using the reporter molecule and inspecting for the presence of the protein of interest.    
   
   
       14 . The method of  claim 13  wherein in step f) the primary antibody has a reporter molecule thereon and step g) is omitted.  
   
   
       15 . The method of  claim 13  wherein the IBM comprises Protein-A or Protein-G.  
   
   
       16 . The method of  claim 13  wherein the IBM is selected by a method that determines binding to immunoglobulins.  
   
   
       17 . The method of  claim 13  wherein the IBM is identified by analysis of materials selected from a library of synthetic compounds, an engineered protein, a compound made in a mammalian system, a polymer made in a non-mammalian system, a compound prepared by recombinant DNA technology, a polymer made by  E. coli  phage display, a protein scaffold library, a peptide display library, a directed evolution library, or a protein array-based library.  
   
   
       18 . The method of  claim 13  wherein the ligand comprises a molecule that occupies the immunoglobulin binding site of an IBM or fragments thereof.  
   
   
       19 . The method of  claim 13  wherein the ligand is isolated from animals.  
   
   
       20 . The method of  claim 19  wherein the ligand comprises pig IgG.  
   
   
       21 . The method of  claim 13  wherein the ligand is selected from the group of compounds consisting of an immunoglobulin, a portion of an immunoglobulin, a fragment of an immunoglobulin, a variant of an immunoglobulin, an engineered protein, a polymer scaffold, an engineered compound, a polypeptide, a compound made in a mammalian system, a polymer made in a non-mammalian system, and a polymer made in an  E. coli  phage display.  
   
   
       22 . The method of  claim 13  wherein in step g) the secondary antibody is responsive only to a selected component of antibody fragments or mixtures thereof or intact antibody molecules.  
   
   
       23 . The method of  claim 12  wherein in step g) the secondary antibody having a reporter molecule is replaced with an IBM having a reporter molecule.  
   
   
       24 . A method for identifying ligands to be used to block immunoglobulin binding molecules and fragments thereof comprising using the IBM that is used in an upstream immunochemical method in a method that identifies molecules that compete for the immunoglobulin binding site on an IBM.  
   
   
       25 . An article of manufacture comprising: 
 packaging material and    a ligand of an immunoglobulin binding molecule.    
   
   
       26 . The article of manufacture of  claim 25  further comprising an immunoglobulin binding molecule.  
   
   
       27 . The article of manufacture of  claim 26  further comprising a solid support for the immunoglobulin binding molecule.  
   
   
       28 . The article of manufacture of  claim 27  further comprising components selected from the group of components consisting of dry solids, primary antibodies, secondary antibodies having a reporter molecule, immunoglobulin binding molecules having a reporter molecule and combinations thereof.  
   
   
       29 . The article of manufacture of  claim 28  wherein a secondary antibody is selected and the secondary antibody recognizes only selected components of a mixture that may contain fragments of antibody, intact antibody, immunoglobulin binding molecule or fragments thereof.  
   
   
       30 . The article of manufacture of  claim 25  further comprising instructions for use.